The overall hypothesis addressed in this project is that the gastrointestinal (Gl) peptide parathyroid hormone-related protein (PTHrP) plays an important regulatory role in the gut, and its dysregulation contributes to disease via aberrant regulation of the phosphatidylinositol-3-kinase (PI3K) pathway. We show that PTHrP increases colon cancer cell xenograft growth in vivo. PTHrP expression is higher in metastatic vs. non-metastatic colon cancer cells, and there is a direct correlation of PTHrP levels with pro-invasive integrin expression and the migratory and invasive potential of these cells in vitro. We find that the PI3K pathway mediates these effects. The focus of this project is to understand the molecular mechanisms via which PTHrP promotes colon cancer cell invasion, by pursuing three Aims. (1) The mechanism(s) governing the upregulation of PI3K by PTHrP will be determined, by asking whether this occurs via an interaction between the PI3K p85<x subunit with an activated PTHrP receptor, and/or is secondary to the PTHrP-mediated upregulation of integrin a6p4. PI3K activity will be measured after exogenous addition of PTHrP to the cell culture, and in cells engineered by stable transfection to overexpress PTHrP. The role of integrins in PI3K activation will be determined using siRNAs to downregulate their expression. (2) The role of downstream effectors regulated by PI3K, including Akt, PTEN, and the small GTP-binding proteins Rac and Cdc42, will be investigated. This aim will be accomplished using such techniques as transfection with dominant-negative mutants, determination of the protein levels by Western immunoblotting and immunocytochemistry, and direct activity measurements. (3) The pathway(s) via which PTHrP upregulates pro-invasive integrin expression will be addressed using Northern blot analysis to detect changes in integrin mRNA levels, pulse-chase analysis to detect alterations in the rate of integrin synthesis and turnover, and FACS analysis to detect changes in integrin intracellular mobilization in PTHrP-overexpressing vs. control colon cancer cells. These studies will allow us to identify the mechanism(s) by which PTHrP promotes invasion and metastases of colon cancer cells, and should ultimately allow the development of new therapeutic strategies aimed at controlling this process.